The ultimate objective of this research project is to identify the molecular mechanism (and/or key mediators) by which osteoclastic resorption is regulated. In this regard, recent studies showed that targeted over-expression of a structurally unique osteoclastic protein-tyrosine phosphatase (PTP-oc) in osteoclastic cells led to a large increase in bone resorption and a marked decrease in bone mass in male but not female transgenic mice, compared to corresponding wild-type littermates. Very recent preliminary studies discovered two novel molecular mechanisms of PTP-oc to regulate the overall osteoclast activity: 1) the EphA4 signaling may function as a negative regulatory mechanism of osteoclastic resorption and PTP-oc stimulates osteoclast differentiation and activity through relieving the inhibitory actions of the EphA4 signaling via dephosphorylation of EphA4; and 2) the PTP-oc signaling increases estrogen receptor (ER) signaling through an upregulation of the c-Src-dependent phosphorylation of ER in osteoclasts, which in turn suppresses bone resorption in adult female but not male PTP-oc transgenic mice. This proposal has three Specific Objectives to investigate these two novel mechanisms of PTP-oc. The first Objective is to demonstrate that the EphA4 signaling is negative regulator of osteoclasts through differential regulation of the various Rho GTPases and through suppression of the Erk1/2-c-Fos-NFAT1c cascade. This will be accomplished through evaluation of the bone phenotype of EphA4-deficient mice in vivo and to determine the effects of deficient EphA4 expression in osteoclast precursors of EphA4 null mice or transgenic over-expression of EphA4 in wild-type precursors on the formation, fusion, adhesion, migration, actin cytoskeleton re-organization, and resorption activity of osteoclasts in vitro. Effects of deficient EphA4 signaling in EphA4 null osteoclasts on the activation states of the various Rho GTPases and the Erk1/2-c-Fos-NFATc1 signaling cascade are also determined. The second Objective is to determine whether PTP-oc enhances by osteoclastic resorption, in part, through suppressing the inhibitory actions of the EphA4 on osteoclasts through direct dephosphorylation of EphA4. This is achieved by confirming that EphA4 is a cellular substrate of PTP-oc, and by demonstrating that over-expression of WT-PTP-oc, but not PD-PTP-oc, would inhibit the suppressive effects of the EphA4 signaling on osteoclast differentiation and activity. It is also tested by showing that the hemotopoietic stem cell-based marrow transplantation with WT- EphA4, but not the mutant lacking the key phosphotyrosine residues, would reverse the osteoporotic phenotype of EphA4 null mice. The third Objective is to determine whether the PTP-oc signaling increases estrogen receptor (ER) signaling through the c-Src-dependent phosphorylation of ER in osteoclasts, which in turn suppresses bone resorption in adult female but not male PTP-oc transgenic mice. This is achieved by a) comparing the bone phenotype and the ER signaling in osteoclasts of pre-pubertal male and female PTP-oc transgenic mice, 2) evaluating the effects of ovariectomy and estrogen replacement on the osteoclastic phenotype of adult female PTP-oc transgenic mice, and 3) evaluating the effects of PTP-oc over-expression in osteoclasts of ER null female mice. Our work should provide important insights into the molecular mechanism of the osteoclast activation and/or the role of a unique osteoclastic enzyme (PTP-oc) in the regulation of two novel signaling mechanisms of osteoclastic resorption. Potential clinical relevance is that aberration in PTP-oc function could be involved in some patients with excess bone resorption and, as such, PTP-oc could be a target for pharmacogenomic treatments of osteoporosis and related bone-wasting diseases.